Characterization of beta‐glucosidase from Physarum polycephalum (LB141)

Abstract

Physarum polycephalum, which is one of the true slime molds, has a unique life cycle that includes plasmodia, sclerotia, spores, and amoebae. Furthermore, the plasmodia cultured in liquid medium form a small lump called microplasmodia. The microplasmodia secrete a large amount of slime that includes the various proteins such as beta‐glucosidase: intracellular soluble, membrane‐bound, and secretory enzymes. Previously, we had reported that the membrane‐bound beta‐glucosidase BglM1, which has a molecular mass of 130 kDa, has a unique primary structure. The structure of BglM1 consists mainly of two regions: the N‐terminal region, which contains a typical glycosyl hydrolase family 3 domains and the C‐terminal region, which has a unique sequence that is unrelated to hydrolytic reactions. Notably, the C‐terminal region of BglM1 contains two calx‐beta motifs known as calcium binding sites, an RGD sequence, and a transmembrane region. In this study, we focus on the characteristics of a secretory enzyme that may be a membrane‐free type of BglM1.The molecular mass of the secretory enzyme purified in the presence of Ca ion and absence of Ca ion is 130 and 95 kDa, respectively. The 130kDa molecules react with antibodies that are specific for the BglM1 N‐terminal and C‐terminal regions. However, the 95kDa molecules are not detected by the C‐terminal antibody. These results indicate that in the absence of Ca ion, the 130kDa molecules are unstable and are degraded to 95kDa molecules. The kinetics data obtained from the 95 and 130kDa molecules showed that the calx region plays an important role on the stability and function of the enzyme. Further, to clarify the physiological functions of BglM1 with a calx region and of the secretory form, we assessed their mRNA expression during the Physarum cell cycle.